molecules Article
An NMR-Guided Screening Method for Selective Fragment Docking and Synthesis of a Warhead Inhibitor Ram B. Khattri 1,† , Daniel L. Morris 1,† , Caroline M. Davis 1 , Stephanie M. Bilinovich 2 , Andrew J. Caras 1 , Matthew J. Panzner 1 , Michael A. Debord 1 and Thomas C. Leeper 1, * 1
2
* †
Department of Chemistry and Biochemistry, The University of Akron, Akron, OH 44325, USA;
[email protected] (R.B.K.);
[email protected] (D.L.M.);
[email protected] (C.M.D.);
[email protected] (A.J.C.);
[email protected] (M.J.P.);
[email protected] (M.A.D.) Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599, USA;
[email protected] Correspondence:
[email protected] or
[email protected]; Tel.: +1-206-708-9546; Fax: +1-330-972-6085 These authors contributed equally to this work.
Academic Editors: Raymond S. Norton and Martin J. Scanlon Received: 29 March 2016; Accepted: 22 June 2016; Published: 16 July 2016
Abstract: Selective hits for the glutaredoxin ortholog of Brucella melitensis are determined using STD NMR and verified by trNOE and 15 N-HSQC titration. The most promising hit, RK207, was docked into the target molecule using a scoring function to compare simulated poses to experimental data. After elucidating possible poses, the hit was further optimized into the lead compound by extension with an electrophilic acrylamide warhead. We believe that focusing on selectivity in this early stage of drug discovery will limit cross-reactivity that might occur with the human ortholog as the lead compound is optimized. Kinetics studies revealed that lead compound 5 modified with an ester group results in higher reactivity than an acrylamide control; however, after modification this compound shows little selectivity for bacterial protein versus the human ortholog. In contrast, hydrolysis of compound 5 to the acid form results in a decrease in the activity of the compound. Together these results suggest that more optimization is warranted for this simple chemical scaffold, and opens the door for discovery of drugs targeted against glutaredoxin proteins—a heretofore untapped reservoir for antibiotic agents. Keywords: glutaredoxin; FBDD; STD; HSQC; trNOE; warhead; ortholog; docking
1. Introduction Fragment-Based Drug Discovery (FBDD) is an emerging method for screening ligands against putative drug target proteins [1]. The goal of the work that follows was applying FBDD techniques to discover and develop a medicinally significant lead molecule with selectivity for a bacterial glutaredoxin while avoiding cross reactivity with the human ortholog. The primary method for discovering prospective leads is a variant of Saturation Transfer Difference (STD) NMR. Positive results were cross-validated via transfer Nuclear Overhauser Effect (trNOE) and Structure Activity Relationship (SAR) by NMR. A library of 463 small fragment compounds was screened for selectivity against two orthologous glutaredoxin proteins, with the rationale that affinity for selective hits may be amplified with chemical synthesis while conserving ortholog specificity. Fragments serve the role of lead molecules to guide development of drug compounds that selectively target the protein of a particular infectious species with reduced likelihood of off-target interactions with host proteins. Although hits obtained from the library are weak (KD > 0.5 mM), they have potential to be chemically
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linked or elaborated to assemble lead compounds with higher affinity. Such an elaboration has been performed, as demonstrated by synthesis of the chimerical compound RK207ACP. Acrylamide moieties have been used to functionalize lead compounds by several researchers to develop irreversible inhibitors for some classes of proteins [2–5]. This acrylamide “warhead” can form a covalent interaction via alkylation with conserved Cys residues in the active sites of these proteins. Although such warheads are often prejudged by medicinal chemists to have poor pharmacokinetics and ADME, recent studies suggest they still have a place in drug development [6]. In particular, linking acrylamide warheads with a specificity-conferring “driving group” can lead to formation of new potent lead compounds [6–8]. We suspect that with certain delivery conditions, e.g., nebulization or topical applications, such warhead derivatives may prove quite useful. Cys residues in the conserved active site of target GRXs are expected to react with acrylamide warheads via a Michael addition reaction, in which the warhead vinyl group is the Michael acceptor and the thiolate group of the active site Cys residue is the Michael donor, driving the formation of a covalent adduct [9]. There are several examples of existing drug discovery campaigns focusing on covalent warheads. Using different electrophilic compounds, many research groups are focused on designing irreversible inhibitors for epidermal growth factor receptor (EGFR) [6,10–12]. These inhibitors were reported to be effective against secondary mutations associated with cancer cells and thus were promoted to clinical trials [11,13,14]. Cocco and coworkers reported that compounds with an α,β-unsaturated electrophilic warhead showed multi-targeting, anti-pyroptotic activities against the cryopyrin inflammasome regulatory pathway [8]. Similarly, Maresso and coworkers, in search for antibiotic agents, found that compounds containing a β-aminoethyl ketone moiety act as irreversible inhibitors against sortase, a surface-protein-modifying enzyme. Furthermore, aryl forms of these compounds were also reported as irreversible inhibitors for several intercellular kinases. A group at the CHDI foundation reported an acrylamide based irreversible inhibitor that showed high potency and specificity for a transglutaminase enzyme [5]. Similarly, Payne and colleagues developed an acrylamide based compound targeting bacterial enoyl-acyl carrier protein reductase (FabI). This compound displayed an MIC value 500 times lower than commercial antibiotics against the Gram-positive bacterium S. aureus [15]. Orthologous glutaredoxin (GRX) proteins from Brucella melitensis (BrmGRX) and Homo sapiens (hGRX) are the target proteins utilized for screening in this study. The National Institute of Allergy and Infectious Diseases (NIAID) classifies B. melitensis as a Category B priority pathogenic organism. B. melitensis is known to cause brucellosis and, while in developed countries Brucella infection rates are relatively low, it is one of the most significant sources of zoonotic airborne bacterial infections in troubled parts of the world including Syria and to a lesser extent Afghanistan [16–18].There are slight differences in amino acid sequences between BrmGRX and hGRX due to evolutionary divergence that result in variations within solvent exposed surface structures of the proteins [19]. This study aims to probe these differences to identify a fragment specific for BrmGRX and to modify that fragment into a lead warhead using an electrophilic group. Our study is focused on developing an irreversible inhibitor for the GRX ortholog of B. melitensis. We chemically synthesized a lead compound containing an acrylamide warhead fused to a fragment hit driving group identified via an NMR-based FBDD assay. The initial fragment hit had selectivity for BrmGRX over hGRX and this subtle selectivity was partially maintained as a warhead functional group was extended into the active site. Selective reaction rates were measured with NMR kinetic assays that determined the modification rates of the GRX orthologs with the control compound (acrylamide) versus ester and acid derivatives of the warhead-fragment chimera. These kinetic data were combined and analyzed to determine the selective reactivity for this new category of irreversible inhibitors. 2. Results and Discussion 2.1. Library This study presents the screening of a small library of fragment compounds against two orthologous proteins using STD NMR [20]. Hits were validated with trNOE [21] and 15 N-HSQC [22]. The library contains 39 scaffolds with different side chains and heteroatoms, the diversity of which
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various in binding affinities for affinities different types of proteins. Parameters such Parameters as partition coefficients (log P), results various binding for different types of proteins. such as partition molecular weight, number of H-bond donors/acceptors the library wereinconstrained be coefficients (log P), and molecular weight, and number of H-bondin donors/acceptors the library to were adherent to the “rule of three” [4].“rule of three” [4]. constrained to be adherent to the 2.2. Library Screening and Hit Validation STD NMR is a popular method for facile identification identification of ligand-protein interactions. Its primary advantage over other ligand screening methods is a reduction in the concentration concentration of protein required for analysis with no size limit for the the target target molecule molecule [20]. [20]. Different Different combinations combinations of of selective selective binders, binders, common binders, and non-binders were obtained from STD screening. Representatives of these sets with relative STD% are shown in Figure 1; a list of fragments fragments is presented in the Supplementary Materials (Figure S1). STD spectra can be collected as separate on and off resonance spectra or as true difference spectra. In this study the former is preferred since they are more convenient for matching peak patterns to reference spectra. The overall overall STD STD hit rate was high, with 21% of the fragments spectra. The interacting with one or both GRXs. The most likely reason for this rate is due to the versatile nature of these small fragments. They They have the potential to fit into multiple binding sites because of their size, leading leadingto toincreased increasedfrequency frequencyofof accommodations [23]. BrmGRX selective hit was rate only was accommodations [23]. TheThe BrmGRX selective hit rate only 2%, whereas the hGRX1 selective found 3%.Therefore, Therefore,a amore morespecific specific subset subset of 2%, whereas the hGRX1 selective raterate waswas found to to bebe 3%. compounds is is proposed proposedto tobind bindselectively selectivelytotoeach each GRX. Venn diagram (Figure 2) compares GRX. AA Venn diagram (Figure 2) compares thethe set set of selective binders, common binders, and non-binders. An example STD spectra of a mixture of of selective binders, common binders, and non-binders. An example STD spectra of a fragments measured against both GRXs GRXs is is shown shown in in Figure Figure 33 (true (true difference differencespectra spectrain inFigure FigureS2). S2). RK246 shows a relative STD percentage (rel. STD%) higher than the threshold value for BrmGRX (Figure 1), but but not not for for hGRX1. hGRX1. Therefore, RK246 is is considered considered aa BrmGRX-selective BrmGRX-selective hit. hit. The STD spectra for RK207 (the final fragment hit chosen for optimization) against BrmGRX is available in Supplementary Figure S7.
Figure different sets sets of of binders. binders. Figure 1. 1. Representative Representative fragments fragments showing showing different
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Figure 2. Venn diagram showingSTD STDresults. results. Fragments with oneone GRXGRX are selective Figure 2. Venn diagram showing Fragmentsthat thatinteract interact with are selective Figure 2. Venn diagram showing STDare results. Fragments that interact with one GRXthat are selective binders. Fragments that bindtotoboth both common binders. are are fragments binders. Fragments that bind are common binders.Non-binders Non-binders fragments do thatnot do not binders. bind to both are common binders. Non-binders are fragments that do not interactFragments with eitherthat protein. interact with either protein. interact with either protein.
Figure 3. On- and off-resonance STD spectra for a fragment pool containing RK246 against two orthologous proteins. (A) The normal 1H-NMR spectrum for the fragment pool; (B) Shows the STD spectra fragment pool with BrmGRX and the STD pool spectra for the fragment withtwo Figure 3. for On-the and off-resonance STD spectra for(C) a is fragment containing RK246 pool against Figure 3. On- and off-resonance STD spectra for a fragment pool containing RK246 against two 1H-NMR hGRX1. These are collected at normal 750 MHz with a cold probe at 0.5 mM fragment and 10 μM protein. spectrum for the fragment pool; (B) Shows the STD orthologous proteins. (A) The 1
orthologous proteins. (A) The normal H-NMR spectrum for the fragment pool; (B) Shows the STD spectra for the fragment pool with BrmGRX and (C) is the STD spectra for the fragment pool with spectra for thehits fragment poolfrom with BrmGRXSTD andscreening (C) is the were STD spectra for the fragment pool with Selective further validated beginning hGRX1. These areobtained collected at 750 primary MHz with a cold probe at 0.5 mM fragment and 10 μM protein. with hGRX1. These are collected at 750 MHz with a cold probe at 0.5 mM fragment and 10 µM protein. transfer Nuclear Overhauser Effect (trNOE) spectroscopy. The principle of trNOE is that transfer of
cross-relaxation protons be measured between were boundfurther and unbound fragments; this with is Selective hitsbetween obtained from can primary STD screening validated beginning influenced by the large correlation time of the ligand-target complex via chemical exchange [21]. Selective hits obtained from primary STD screening were further validated beginning transfer Nuclear Overhauser Effect (trNOE) spectroscopy. The principle of trNOE is that transfer with of Upon ligand binding to protons protein, a (trNOE) large negative NOE in trNOE spectrum isofobserved [20]. 6%this of is transfer Nuclear Overhauser Effectcan spectroscopy. The principle trNOE is that transfer cross-relaxation between be measured between bound and unbound fragments; STD-selective hits for both GRX orthologs were found to show negative NOE peaks in the trNOE by thebetween large correlation timebeofmeasured the ligand-target via chemical [21]. of influenced cross-relaxation protons can betweencomplex bound and unboundexchange fragments; this spectra, thus doubly confirming some of the STD selective hits to be unique binders with their Upon ligand binding to protein, a large negative NOE in trNOE spectrum is observed [20]. 6% of is influenced by the large correlation time of the ligand-target complex via chemical exchange [21]. respective GRX ortholog. Mixtures of hit fragments were used in order to reduce the number of STD-selective hits fortoboth GRX aorthologs were found to negative NOE in the trNOE Upon ligand binding protein, large in show trNOE is peaks observed [20]. 6% of spectra collected (both to conserve NMRnegative time and NOE d8 glycerol) and tospectrum allow for possible observation spectra, thushits doubly confirming some of were the STD selective hitsnegative to be unique binders with their STD-selective for both GRX orthologs found to show NOE peaks in the trNOE of interligand NOEs (iLOEs) useful for the process of linking and merging fagments. These were not respective ortholog. Mixtures hit were in to order to reduce the number of spectra, thusGRX doubly confirming of fragments the STDinselective hits be unique binders with their considered in the present systemsome butofcould be used futureused fragment growing/linking campaigns. spectra collected (both to conserve NMR time and d 8 glycerol) and to allow for possible observation About GRX 55% ortholog. of BrmGRXMixtures selective ofhits using used STD in were confirmed using respective hit determined fragments were order to reduce thetrNOE. number of of Comparison interligand NOEs (iLOEs) useful for the process ofagainst linking andGRXs merging fagments. These were trNOE for fragment mixtures both are shown in Figure 4, wherenot spectra collectedof(both tospectra conserve NMR time and d glycerol) and to allow for possible observation 8 considered in the present system but could be used in future fragment growing/linking campaigns. of interligand NOEs (iLOEs) useful for the process of linking and merging fagments. These were not About 55% of BrmGRX selective hits determined using STD were confirmed using trNOE. considered in the present system but could be used in future fragment growing/linking campaigns. Comparison of trNOE spectra for fragment mixtures against both GRXs are shown in Figure 4, where About 55% of BrmGRX selective hits determined using STD were confirmed using trNOE. Comparison
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of trNOE spectra for fragment mixtures against both GRXs are shown in Figure 4, where fragments RK207, RK214 and RK246 found were to be found selective forselective BrmGRX. didRK155 not bind either fragments RK207, RK214were and RK246 to be forRK155 BrmGRX. didto not bindGRX to 15 ortholog and was thus considered a “non-hit.” Overlays of the N-HSQC spectra for RK155, RK214, 15 either GRX ortholog and was thus considered a “non-hit.” Overlays of the N-HSQC spectra for RK207 andRK214, RK246RK207 against BrmGRX are shown in Supplementary Figure S3. RK155, and RK246 against BrmGRX are shown in Supplementary Figure S3.
Figure 4. Comparison of the trNOE spectra between the mixture of 0.5mM each of RK207, RK246, Figure 4. Comparison of the trNOE spectra between the mixture of 0.5mM each of RK207, RK246, RK214, and RK155 with: (A) 10 μM of BrmGRX; (B) 10 μM of hGRX1. The sign of cross-peaks from RK214, and RK155 with: (A) 10 µM of BrmGRX; (B) 10 µM of hGRX1. The sign of cross-peaks from bound fragments are the same sign to that of the protein (the sign of the diagonal), thus confirming bound fragments are the same sign to that of the protein (the sign of the diagonal), thus confirming binding. Peaks corresponding to respective fragment protons are assigned with the same numbers. binding. Peaks corresponding to respective fragment protons are assigned with the same numbers. iLOEs present between fragments as a possible indication of synergistic binding that could be used iLOEs present between fragments as a possible indication of synergistic binding that could be used in in future fragment linking studies. Unassigned protons were not picked due to significant signal future fragment linking studies. Unassigned protons were not picked due to significant signal overlap overlap or failing to appear in the spectrum. Red, blue and purple dotted oval circles on the left or failing to appear in the spectrum. Red, blue and purple dotted oval circles on the left spectrum spectrum represent peaks for RK207, RK246, and RK214, respectively. As above these spectra are represent peaks for RK207, RK246, and RK214, respectively. As above these spectra are collected at collected at 750 MHz with cold probe. 750 MHz with cold probe.
Similarly, the entire set of positive hits, including common hits, from STD and trNOE primary screening were with 15N-HSQC [24]. Among thehits, 99 hits, only 27%and induced chemical Similarly, thefurther entirevalidated set of positive hits, including common from STD trNOE primary 15 shift perturbation (CSP) of protein peaks when used in excess concentration (5to 20-fold). Mapping screening were further validated with N-HSQC [24]. Among the 99 hits, only 27% induced chemical shifted residues to structure surfaces revealed bind concentration in the shallow groove present near the shift perturbation (CSP) of protein peaks whenmost usedhits in to excess (5- to 20-fold). Mapping activeresidues site of GRXs identified previously in a comparative analysis [19]. Figurepresent 5 displays thethe shifted to structure surfaces revealed most hits tostructural bind in the shallow groove near binding pockets occupied by RK207 defined by CSPs upon binding with BrmGRX and hGRX1. RK207 active site of GRXs identified previously in a comparative structural analysis [19]. Figure 5 displays in the previously described pocket defined adjacent by to the conserved CPYC active of BrmGRX thebinds binding pockets occupied by RK207 CSPs upon binding withsite BrmGRX and when hGRX1. observed at or below a stoichiometric concentration of ligand to protein. However, when an excess RK207 binds in the previously described pocket adjacent to the conserved CPYC active site of BrmGRX of RK207 was used the specificity was lost and the compound further perturbed additional residues when observed at or below a stoichiometric concentration of ligand to protein. However, when an in non-specific interactions. Evidence for such interactions is displayed in Supplementary Figure S4. excess of RK207 was used the specificity was lost and the compound further perturbed additional These results clearly suggest that 1D NMR, especially STD NMR, is more sensitive than 2D NMR in residues in non-specific interactions. Evidence for such interactions is displayed in Supplementary the detection of weak fragment interactions with target proteins. In addition, selectivity of some Figure S4. These results clearly suggest that 1D NMR, especially STD NMR, is more sensitive than 2D compounds identified during the STD screen were masked at the higher concentrations used for NMR in the detection of ofthe weak fragment interactions proteins. In addition, selectivity HSQC. Dissociation fragment during the mixingwith timetarget in trNOE may lead to signal loss, making of some identified the NMR STD screen were masked the higher concentrations used thiscompounds technique less sensitiveduring than STD [25]. Similarly, lack ofatstrong ring current effects from forsome HSQC. Dissociation of the fragment during the mixing time in trNOE may lead to signal loss, 15 fragments may cause low sensitivity observed in N-HSQC experiments [24,26]. For these making this technique less sensitive than STD NMR [25]. Similarly, lack of strong ring current effects reasons, we strongly favor STD as the primary screening method for identifying fragment ligands. from some fragments may cause low sensitivity observed in 15 N-HSQC experiments [24,26]. For these reasons, we strongly favor STD as the primary screening method for identifying fragment ligands.
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Figure 5. Comparison of binding pockets defined by compounds based on CSPs for BrmGRX and hGRX1.
Figure 5. Comparison of binding pockets defined by compounds based on CSPs for BrmGRX and Surface and secondary structures for: defined (A,B) RK207 vs. hGRX1, defining multiple possibleand binding Figure 5. Comparison of binding pockets compounds based on CSPs for BrmGRX hGRX1. Surface and secondary structures for: by (A,B) RK207 vs. hGRX1, defining multiplehGRX1. possible sites; (C,D) RK207 vs. BrmGRX, having binding pockets near to the conserved CPYS active site; (E,F) Surface and(C,D) secondary structures for: (A,B) RK207 vs. hGRX1, defining multiple possible binding binding sites; RK207 vs. BrmGRX, having binding pockets near to the conserved CPYS active RK207ACPacid [6] vs BrmGRX; (G,H) RK207ACPester [5] vs. BrmGRX; (I,J) Acrylamide vs. BrmGRX, sites; (C,D) RK207 vs. BrmGRX, having binding pockets near to the conserved CPYS active site; (E,F) site; (E,F) RK207ACPacid [6] vs BrmGRX; RK207ACPester vs.CSPs BrmGRX; (I,J)the Acrylamide respectively (Patches: yellow-CPYC active(G,H) site; light blue—residues[5] with exceeding standard vs. RK207ACPacid [6] vs(Patches: BrmGRX;yellow-CPYC (G,H) RK207ACPester [5]light vs. BrmGRX; (I,J) Acrylamide BrmGRX,the BrmGRX, respectively site; blue—residues with CSPsvs. exceeding deviation; dark blue—residues exceeding 2× active the standard deviation). respectively (Patches: yellow-CPYC active site; light blue—residues with CSPs exceeding the standard standard deviation; dark blue—residues exceeding 2ˆ the standard deviation). exceeding 2× the standard deviation). 2.3.deviation; Selection dark of Hitblue—residues for Optimization
2.3.2.3. Selection of of Hit forforOptimization Based onHit NMR screening results, RK207 was chosen for further study. In spite of its small relSTD%, Selection Optimization which is just barely above the threshold for a positive hit, the fragment’s dissociation constant (Kd)
Based onon NMR screening results, RK207 was chosen forfurther further study.InInspite spite its small relSTD%, NMR results, RK207 wasselective chosen for ofof its small relSTD%, forBased BrmGRX is 5 toscreening 10-fold better than all other hits found.study. Kd values for the best fragments, which is just barely above the threshold for a positive hit, the fragment’s dissociation constant (K ) which is justRK207 barelyand above the were threshold for a positive hit, the fragment’s including RK246, determined using 15N-HSQC (Figure 6). dissociation constant (Kd) d forfor BrmGRX is 5 to 10-fold better than all other selective hits found. K values for the best fragments, BrmGRX is 5 to 10-fold better than all other selective hits found. Kddvalues for the best fragments, 15 N-HSQC (Figure 6). including RK207 and RK246, including RK207 and RK246,were weredetermined determinedusing using 15N-HSQC (Figure 6).
Figure 6. Determination of dissociation constant for ligand-protein complex using 2D NMR. As above these spectra are collected at 750 MHz with a cold probe (A) 15N-HSQC titration of fragment RK207 against BrmGRX. The red contour is apo, blue is 0.2 mM fragment, purple is 0.5 mM, cyan is 2 mM,
Figure Determinationofofdissociation dissociation constant for for ligand-protein complex using 2D NMR. AsAs above Figure 6. 6. Determination ligand-protein complex magenta is 5.2 mM, and yellow is 6 mMconstant As above these spectra are collected at 750using MHz 2D withNMR. cold probe;above 15 these spectra collected 750MHz MHzresidues withaacold coldwere probe (A)to titration of fragment RK207 15N-HSQC these spectra areare collected atatperturbed 750 with probe (A) N-HSQC titration RK207 (B) An expanded view of that used calculate K d; (C) Bestof fitfragment dissociation against BrmGRX. The red contour is apo, blue is 0.2 mM fragment, purple is 0.5 mM, cyan is 2is mM, against BrmGRX. The red contour is apo, blueCurves is 0.2 for mM fragment, purple 0.573L mM, 2 mM, constant curves obtained from CSP analysis. residues 70S, 71D, 72Disand arecyan shown. magenta is 5.2 mM, and yellowisis66mM mMAs Asabove abovethese these spectra spectra are with cold probe; magenta is 5.2 mM, and yellow arecollected collectedatat750 750MHz MHz with cold probe; RK207 was found to be ~2.5-foldresidues more reactive towards than hGRX1, with Kd values An expanded view perturbed residues that were were usedBrmGRX to calculate calculate KKdfor ; ;(C) Best fitfit dissociation (B)(B) An expanded view ofof perturbed that used to (C) Best dissociation d of constant 0.98 ± curves 0.24 and 2.45 ± 0.52 mM respectively. However, no significant was observed curves obtained from CSP analysis.Curves Curves for residues residues 70S, 72D are shown. constant obtained from CSP analysis. for 70S, 71D, 71D,selectivity 72Dand and73L 73L are shown.in RK246 for the BrmGRX ortholog on the basis of HSQC-monitored Kd (10.63 ± 5.18 mM for BrmGRX and 5.22 ±was 1.16 found mM fortohGRX1) in spite of itsreactive larger apparent effects.than Several binding with experiments RK207 be ~2.5-fold more towardsSTD BrmGRX for hGRX1, Kd values RK207 was found to be ~2.5-fold more towards BrmGRX than The for hGRX1, with Kd values N-HSQC-NMR forreactive RK207 analogs against BrmGRX. goal of the post-hoc of were 0.98 ±performed 0.24 and using 2.45 ±150.52 mM respectively. However, no significant selectivity was observed in of 0.98 ˘ 0.24 and 2.45 ˘ 0.52 mM respectively. However, no significant selectivity was observed in analysis scaffold class was to orHSQC-monitored how the carboxylic acid moiety± of RK207 RK246 for of thethe BrmGRX ortholog ondetermine the basisifof Kd (10.63 5.18 mMcontributed for BrmGRX RK246 for the BrmGRX ortholog on the basis of HSQC-monitored K (10.63 ˘ 5.18 mM for BrmGRX to5.22 these± interactions. shownin in spite Figure RK021, contains amide group, shows weaker CSPs d Several and 1.16 mM forAs hGRX1) of7,its largerwhich apparent STDan effects. binding experiments
andwere 5.22performed ˘ 1.16 mMusing for hGRX1) in spite offor itsRK207 larger analogs apparent STD effects. Several binding experiments 15N-HSQC-NMR against BrmGRX. The goal of the post-hoc 15 N-HSQC-NMR for RK207 analogs against BrmGRX. The goal of the post-hoc were performed using analysis of the scaffold class was to determine if or how the carboxylic acid moiety of RK207 contributed analysis ofinteractions. the scaffoldAs class was in to Figure determine if or how the carboxylic acid moiety of RK207 contributed to these shown 7, RK021, which contains an amide group, shows weaker CSPs
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Molecules 2016, 21, 846 7 of 22 to these interactions. As shown in Figure 7, RK021, which contains an amide group, shows weaker CSPsMolecules than the same fragment with a hydroxyl group (RK157). Large CSPs were observed for fragments 21, 846 7 of 22 than the2016, same fragment with a hydroxyl group (RK157). Large CSPs were observed for fragments containing a carboxyl group, indicating its importance in binding. It was also observed containing a carboxyl group, indicating its importance in binding. It was also observed that that the the than theofsame fragmentgroups with a plays hydroxyl group (RK157). Large CSPs were observed for fragments orientation functional an important role in binding. The significance of carboxyl orientation of functional groups plays an important role in binding. The significance of carboxyl containing a carboxyl group, indicating its importance in binding. It was also observed that the groups in binding affinity within different proteins hasbeen been previously elucidated [27,28]. groups in binding affinity within differentclasses classes of of proteins has previously elucidated [27,28].
orientation of functional groups plays an important role in binding. The significance of carboxyl groups in binding affinity within different classes of proteins has been previously elucidated [27,28].
Figure 7. 15 (A) N-HSQC comparisonof ofbinding binding modes functional groups as well Figure 7. (A) N-HSQC comparison modesamong amongdifferent different functional groups as as well as 15N their orientation. 2 mM of RK207 and each of its analog fragments are observed against 0.25 mM 15 N their Figure orientation. mM of RK207 and each of its modes analogamong fragments are functional observed groups againstas0.25 7. (A) 152N-HSQC comparison of binding different wellmM as BrmGRX; (B) Enlarged S70 peaks; and (C) RK207 and its’ analogs. (Red: apo, blue: RK207, brown BrmGRX; (B) Enlarged S70 andeach (C) of RK207 and fragments its’ analogs. (Red: apo, blue: RK207, brown their orientation. 2 mM ofpeaks; RK207 and its analog are observed against 0.25 mM 15N RK192, black: RK445, magenta: RK157,green: green: RK021, RK021, cyan: RK054). RK192, black: RK445, magenta: RK157, cyan: RK054). BrmGRX; (B) Enlarged S70 peaks; and (C) RK207 and its’ analogs. (Red: apo, blue: RK207, brown 15
RK192, black: RK445, RK157, green: RK021, cyan: RK054). 2.4. Fragment Docking formagenta: Lead Development
2.4. Fragment Docking for Lead Development
Using molecular dynamics (MD) simulations, computational CSP calculations, and a final STD 2.4. Fragment Docking for Lead Development Using molecular dynamics (MD) was simulations, CSP calculations, and a final STD filter, RK207’s carboxylic acid moiety predicted computational to be oriented towards the active site of BrmGRX. Usingcarboxylic molecular dynamics (MD) simulations, computational CSP calculations, and site a final STD filter,MD RK207’s moiety predicted to be oriented towards theand active of of BrmGRX. simulations were acid carried out inwas Molecular Operating Environment (MOE) a database 48 filter, RK207’s carboxylic acid moiety was predicted to be oriented towards the active site of BrmGRX. MD simulations were carried ingenerated Molecular Environment a database possible RK207 docked posesout were [29].Operating (Figure 8A). Each pose and(MOE) the apo and structure were of MD simulations were carried out in Molecular Operating Environment (MOE) and a database of 48 then runRK207 through SHIFTSposes 5.0.1 and computational CSPs(Figure were calculated for backbone protons 48 possible docked were generated [29]. 8A). Each pose andamide the apo structure possible RK207 docked poses were generated [29]. (Figure 8A). Each pose and the apo structure were [30]. Predicted shifts were scored against experimental shifts, where computational shifts of the pose werethen thenrun run through SHIFTS 5.0.1 and computational CSPs were calculated for backbone amide through SHIFTS 5.0.1 and computational CSPs were calculated for backbone amide protons that[30]. agree most withshifts experimental data against is indicative of the poseshifts, beingwhere measured experimentally. protons Predicted were scored experimental computational shifts [30]. Predicted shifts were scored against experimental shifts, where computational shifts of the poseof the pose that thatagree agreemost most with experimental is indicative thebeing posemeasured being measured experimentally. with experimental datadata is indicative of the of pose experimentally.
Figure 8. NMR guided docking of RK207. (A) Initial docking of 49 possible non-duplicate poses. The BrmGRX surface is rendered in grey; (B) Experimental proton shift perturbations. A standard deviation Figure 8. NMR guided docking of RK207. (A) Initial docking of 49 possible non-duplicate poses. The of 8. 0.006 is observed. and computational below thresholdnon-duplicate were set to zeroposes. Figure NMR guided Experimental docking of RK207. (A) Initial shifts docking of this 49 possible BrmGRX surface is rendered in grey; (B) Experimental proton shift perturbations. A standard deviation before scoring; (C) is Computational CSP filtering resulting in proton these topshift 5 poses; (D) Group epitope The BrmGRX surface rendered in grey; (B) Experimental perturbations. A standard of 0.006 is observed. Experimental and computational shifts below this threshold were set to zero mapping comparison of CSP filtered poses reveals a single pose in agreement with STD data. Thewere deviation of 0.006 is observed. Experimental and computational shifts below this threshold before scoring; (C) Computational CSP filtering resulting in these top 5 poses; (D) Group epitope protein surfacescoring; is rendered with atom color. CSP filtering resulting in these top 5 poses; (D) Group set tomapping zero before Computational comparison of(C) CSP filtered poses reveals a single pose in agreement with STD data. The epitope mapping of CSP filtered protein surfacecomparison is rendered with atom color. poses reveals a single pose in agreement with STD data.
The protein surface is rendered with atom color.
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Computational prediction of proton chemical shifts has not been routinely used in the past for FBDD projects. McCoy and Wyss first used this method with SHIFTS in 2000 to accurately predict the orientation of a tryptophan-like inhibitor for calmodulin [31,32]. They would later go on to use Molecules 2016, 21, 846 8 of 22 the same approach in the development of an inhibitor for a hepatitis C protease [33]. Filtering docked Computational prediction proton chemical shifts has not been proton routinely usedtoincomputationally the past for poses for fragments on the bases ofof comparing experimental backbone shifts FBDD projects. McCoy and Wyss first used this method with SHIFTS in 2000 to accurately generated shifts using a Pscore was only recently validated for FBDD-type screening projects predict by Aguirre the orientation of a tryptophan-like inhibitor for calmodulin Theytowould later go onmost to useaccurate the et al. over several manuscripts [34–36]. We employed these[31,32]. methods determine the same approach in the development of an inhibitor for a hepatitis C protease [33]. Filtering docked poses binding pose of RK207. for fragments on the bases of comparing experimental backbone proton shifts to computationally Initial scoring with the CSP filter and using an arbitrary Pscore threshold of 0.017 resulted in a generated shifts using a Pscore was only recently validated for FBDD-type screening projects by cluster of the top 5 RK207 poses bound near the α3 helix of BrmGRX that lies adjacent to conserved Aguirre et al. over several manuscripts [34–36]. We employed these methods to determine the most CPYCaccurate active binding site. (Figure 8C) Rel STD% from RK207 STD experiments were used to generate pose of RK207. group epitope mapping of the ligand, agreed with only onethreshold pose in of this cluster (Figure Initial scoring with the CSP filterwhich and using an arbitrary Pscore 0.017 resulted in a 8D) .The resulting final pose is primarily stabilized with the double bonded oxygen of the carboxylic cluster of the top 5 RK207 poses bound near the α3 helix of BrmGRX that lies adjacent to conserved CPYC acid (Figure Relthe STD% from RK207 STD were used to generate group epitope groupactive in a site. polar bond8C) with backbone amide ofexperiments S70 and the pyrrole ring in a CH–π interaction mapping the ligand, which agreed with onlycontributions one pose in thisto cluster (Figure 8D) .Thealso resulting final with an Hγ ofof R54. Other residues with minor binding that may be conferring pose is primarily stabilized with the double bonded oxygen of the carboxylic acid group in a polar bond species selectivity include the sidechains of Y18, S52, F57, P58, G69 and the backbone amide of D71. the backbone S70 and the pyrrole ring in a acid CH–πmoiety interaction with an Hγ of R54. Other site, After with examining these amide resultsofconfirming the carboxylic oriented toward the active residues with minor contributions to binding that may also be conferring species selectivity include chemical elaboration was considered for growing the compound in that direction. Two synthetic routes the sidechains of Y18, S52, F57, P58, G69 and the backbone amide of D71. After examining these results were considered for optimization. The first route comprised linking this fragment with a ruthenium confirming the carboxylic acid moiety oriented toward the active site, chemical elaboration was piano-stool compound and the second route consisted of coupling with routes an acrylamide moiety.for considered for growing the compound in that direction. Two synthetic were considered optimization. The first route comprised linking this fragment with a ruthenium piano-stool compound
2.5. Initial Warhead-Fragment Strategy: Ruthenium Arene Derivatives and the second route consisted of coupling with an acrylamide moiety.
The first attempt to couple a warhead to RK207 by adding a ruthenium piano-stool moiety 2.5. Initial Warhead-Fragment Strategy: Ruthenium Arene Derivatives was performed simultaneous to the experiments used for the post-hoc analysis described above. Initial, and somewhat preliminary, studies the carboxylate RK207 The first attempt to couple a docking warhead to RK207suggested by adding athat ruthenium piano-stoolgroup moietyofwas mightperformed be pointing toward the active site. This seemed like a convenient coupling simultaneous to the experiments used carboxylate for the post-hoc analysis described above. Initial, groupand and in the sequence of experiments collected, analysis hadofnot yetmight determined somewhat preliminary, docking studies suggestedthe thatpost-hoc the carboxylate group RK207 be toward the active site. ThisThe carboxylate seemed like a convenient coupling group and in the that itpointing was required for binding. initial strategy was to couple a ruthenium piano-stool sequence of experiments collected, the analysis hadan notorganometallic-based yet determined that it was compound derived from benzylamine to post-hoc RK207, generating leadrequired compound. for binding. The initial strategy waspopular to coupleinaanti-cancer ruthenium piano-stool compound deriveddesign, from as Ru-containing compounds have been and antibiotic therapeutic benzylamine to RK207, generating an organometallic-based lead compound. Ru-containing compounds reviewed recently [37,38]. These compounds also tend to interact with soft nucleophilic centers in have been popular in anti-cancer and antibiotic therapeutic design, as reviewed recently [37,38]. proteins, such as the thiolates present in GRX active sites [39]. Ru-containing complexes were therefore These compounds also tend to interact with soft nucleophilic centers in proteins, such as the thiolates investigated in their ability to bind to GRX. Early attempts included a complex containing both Ru(III) present in GRX active sites [39]. Ru-containing complexes were therefore investigated in their ability and RK207. Compound (Figure included 9) is an example such a complex; however, the carboxyl group to bind to GRX. Early3attempts a complexofcontaining both Ru(III) and RK207. Compound 3 is involved in an amide bond, rather than being a free moiety. The synthesis scheme for compound (Figure 9) is an example of such a complex; however, the carboxyl group is involved in an amide bond, 3 is shown in Supplementary Scheme Interactions of thefor complex with3BrmGRX observed using rather than being a free moiety.S1. The synthesis scheme compound is shown were in Supplementary 15 N-HSQC, 15N-HSQC, where it was Schemewhere S1. Interactions of the complex with BrmGRX were it was discovered that the RK207-Ru dimerobserved complexusing did not bind to BrmGRX under discovered that the RK207-Ru dimer complex did not bind BrmGRX under ambient temperatures. ambient temperatures. Lack of binding was likely due to to loss of a salt bridge formed between the Lack of binding was likely due to loss of a salt bridge formed between the carboxylate moiety of RK207 carboxylate moiety of RK207 and one of the basic residues in BrmGRX, probably R54. and one of the basic residues in BrmGRX, probably R54.
Figure 9. Structures compounds. Figure 9. Structuresfor forsome some synthesized synthesized compounds.
Similarly compound 4, was also found to be ineffective against BrmGRX when monitored via Similarly compound 4, was also found to be ineffective against BrmGRX when monitored via 15N-HSQC because of the unavailability of the carboxyl group to participate at zero NMR time. 15 N-HSQC because of the unavailability of the carboxyl group to participate at zero NMR time. Compound 7, which has an ester group, also did not bind with BrmGRX; however, compound 8, the Compound 7, which has an ester group, also did not bind with BrmGRX; however, compound 8, the
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hydrolyzed acid analog of compound 7 was found to bind to BrmGRX at the zero NMR time. Structure Molecules 2016, 21, 846 9 of 22 for these compounds is shown in Figure 9 and the synthesis schemes for compound 7 and 8 are shown in schemes 2 and 3acid of analog the synthesis portion materials methods part, hydrolyzed of compound 7 wasoffound to bindand to BrmGRX at the zerorespectively NMR time. Structure for these compounds is shown in Figure 9 and synthesis schemes compound 7 and 8itare shown Upon further elucidation of the role of thethe carboxyl moiety infor binding to GRX, was discovered in schemes charged 2 and 3 ofcarboxyl the synthesis portion of materials and methods part, respectively that a negatively group is required. When binding is observed at a lower pH of 4.5 Upon further elucidation of the role of the carboxyl moiety in binding to GRX, it was discovered (just below the pKa value of carboxylic acid), fragments such as RK207, RK445, and RK192 do not bind that a negatively charged carboxyl group is required. When binding is observed at a lower pH of 4.5 with BrmGRX. This at loweracid), pH fragments values, the carboxyl moiety these fragments becomes (just below thesuggests pKa valuethat of carboxylic such as RK207, RK445,in and RK192 do not bind protonated losesThis its binding suggesting modifications the carboxyl moiety may be withand BrmGRX. suggests capacity, that at lower pH values,that the carboxyl moiety inatthese fragments becomes protonated and loses its binding capacity, suggesting that modifications at the carboxyl moiety may deleterious to affinity. be deleterious to affinity.
2.6. Subsequent Warhead-Fragment Strategy: Acrylamide Derivatives of a Modified Fragment 2.6. Subsequent Warhead-Fragment Strategy: Acrylamide Derivatives of a Modified Fragment
Once the importance of the carboxyl group for binding was established, RK207 was modified Once the importance of the carboxyl group for binding was established, RK207 was modified anotheranother way toway produce a lead molecule with a preserved carboxyl group and a cysteine-alkylating to produce a lead molecule with a preserved carboxyl group and a cysteine-alkylating acrylamide warhead [10]. Another RK207notnot in the cluster, but 13th ranked 13th acrylamide warhead [10]. Anotherpose pose of of RK207 in the top-5top-5 cluster, but ranked of the 48 of the 48 original poses postpost CSP filtering inthe theopposite opposite direction ofwinning the winning in 8D Figure 8D original poses CSP filteringisisoriented oriented in direction of the pose inpose Figure andnext is thehighest next highest scoring pose that agreesmost most with epitope mapping data (Figure 10). and is the scoring pose that agrees withgroup group epitope mapping data (Figure 10).
Figure 10. Second ranking pose after CSP and STD filter. The protein surface defining the binding site
Figure 10. Secondwith ranking is rendered atom pose color. after CSP and STD filter. The protein surface defining the binding site is rendered with atom color. This pose has RK207’s pyrrole ring oriented towards the active site with the carboxylic acid moiety stabilized by polar interactions with R54’s guanidinium group. Other stabilizing residues include This pose has RK207’s pyrrole ring oriented towards the active site with the carboxylic acid moiety sidechains of Y18, S52, F57, P58 and the backbone amide of D71; all of the same residues stabilizing the stabilized by polar interactions with R54’s guanidinium group. Other stabilizing residues include best pose save for G69. At this point it was hypothesized that multiple binding modes may be possible sidechains Y18, S52, the backbone amide of D71;[40]. all of same residues stabilizing the andof indeed may F57, even P58 be a and consequence of species selectivity Thethe addition of an acrylamide best pose save for this point it was that multiple binding modesacrylamide may be possible warhead offG69. of theAt pyrrole ring could stillhypothesized result in a higher affinity binder versus naked by increasing residency time in the binding pocket conferred by the RK207 group The and indeed may even be a consequence of species selectivity [40]. Thedriving addition of [41]. an acrylamide warhead covalent inhibitors only requires they affinity stay in the binding pocketnaked for just enough warheadnature off ofofthe pyrrole ring could still result in athat higher binder versus acrylamide by time for the warhead to react with a target residue before becoming permanently fixed. Despite the increasing residency time in the binding pocket conferred by the RK207 driving group [41]. The nature sub-optimal pose, an acrylamide warhead off of RK207 pyrrole ring could shift the binding preference of warhead inhibitors requires that they stay in the binding pocket for just enough of thecovalent RK207 driving group toonly this lower ranked pose. time for theIn warhead to react with a target residue before becoming permanently Despite the the first step, an amine group was introduced to RK207 via copper catalyzed fixed. N-arylation sub-optimal pose, an acrylamide warhead off of 4-iodobenzoate RK207 pyrroleinring could shift the binding preference of (1H-pyrrol-3-yl) methanamine with methyl order to facilitate further coupling with acrylic acidgroup (Scheme [42]. This coupling of the RK207 driving to1)this lower ranked reaction pose. results in the formation of precursor 4 with 20% first yield step, as a white powder.group In the second step of the reaction, compound 4 was further coupled with In the an amine was introduced to RK207 via copper catalyzed N-arylation of acrylic acid using the standard amide coupling agents HATU and trimethylamine [43]. This reaction (1H-pyrrol-3-yl) methanamine with methyl 4-iodobenzoate in order to facilitate further coupling with resulted in the formation of compound 5 as a yellowish solid with 23% yield. Finally, compound 5 acrylic acid (Scheme 1) [42]. Thisbase coupling results incompound the formation precursor 4 with 20% was hydrolyzed with strong to affordreaction the carboxylic acid 6 with of 63% yield.
yield as a white powder. In the second step of the reaction, compound 4 was further coupled with acrylic acid using the standard amide coupling agents HATU and trimethylamine [43]. This reaction resulted in the formation of compound 5 as a yellowish solid with 23% yield. Finally, compound 5 was hydrolyzed with strong base to afford the carboxylic acid compound 6 with 63% yield.
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Scheme 1. Syntheticscheme schemefor for RK207ACP RK207ACP lead Scheme 1. Synthetic leadcompounds. compounds.
2.7. Kinetic Studies and Binding Sites Preferred by Acrylamide Warhead Containing Inhibitor
2.7. Kinetic Studies and Binding Sites Preferred by Acrylamide Warhead Containing Inhibitor Kinetic studies were performed with a five-fold excess of compound 5 and compound 6 against 15N BrmGRX Kinetic were performed withRate a five-fold excesscomplex of compound 5 and against reducedstudies via 15N-HSQC. of inhibited formation wascompound inferred by6 the 15 N BrmGRX via 15 N-HSQC. Rate of inhibited complex formation was inferred by the reduced broadening rate of active site cysteine peaks. Compound 5 showed promising results, taking less than 24 h forrate the reaction to run completion (Supplementary S5A). At the zero time-point, there less broadening of active site to cysteine peaks. CompoundFigure 5 showed promising results, taking were no the CSPs observed for any residues. After an hour of incubation at 25At °Cthe many than 24 h for reaction to run to completion (Supplementary Figure S5A). zeroresidues, time-point, the three spectrally visible active site peaks, and/or broadening. of these there including were no CSPs observed for any residues. After showed an hourCSPs of incubation at 25 ˝ CMost many residues, residues lie between the α1 helix and β2 sheet of BrmGRX, near the conserved CPYC active site. These including the three spectrally visible active site peaks, showed CSPs and/or broadening. Most of data suggest that the warhead functional group is reacting with the active site, which produces effects on these residues lie between the α1 helix and β2 sheet of BrmGRX, near the conserved CPYC active site. nearby residues. Compound 5 was also found to perturb residues in the α2 and α3 helices (Figure 5). The Thesedriving data suggest that the warhead functional group is reacting with the active site, which produces group (the ester form of RK207) accelerates the rate of reaction. Under the same experimental effectsconditions on nearby residues. Compound 5 was alsoof found to perturb residues in thethan α2 ten anddays α3 helices a reaction containing five-fold excess unlinked acrylamide took more to (Figure 5). The driving group (the ester form of RK207) accelerates the rate of reaction. Under the same reach completion. experimental conditions a reaction containing five-fold oftime-point, unlinked acrylamide took A similar study was performed for compound 6. Atexcess the zero many residues thatmore were than associated with the unmodified RK207 binding site showed CSPs, but no changes were observed for ten days to reach completion. active study site residues. Additionalfor residues started6.toAt show chemical shift changes as well Athe similar was performed compound the HSQC zero time-point, many residues thataswere broadening effects after 72 h of incubation at room temperature (Supplementary Figure S5B). From associated with the unmodified RK207 binding site showed CSPs, but no changes were observed for CSPs mapping obtained from 15N-HSQC experiment, compound 6 was found to bind in the opposite the active site residues. Additional residues started to show HSQC chemical shift changes as well as direction to that of compound 5, between α1 and α3 helixes near the active site (Figure 5). The reaction broadening effects after 72 h of incubation at room temperature (Supplementary Figure S5B). From runs to completion between15168 to 192 h of incubation at 25 °C. CSPs mapping obtained from N-HSQC experiment, compound 6 was found to bind in the opposite Compound 6 was found to be less effective than its ester form. After scrutinizing the CSP map direction to that compound 5, between α1this and α3 helixes near of theanactive site (Figurethe 5).carboxylic The reaction (Figure 5E,F)ofthe most probable reason for might be formation H-bond between ˝ runs to completion 168 to 192compound h of incubation atthe 25 primary C. acid and S70 or between R54, resulting in the favoring binding pose oriented in such a Compound was found to be away less effective than its form. After scrutinizing the CSP way that the 6 warhead is directed from the active siteester and remains unavailable for binding with map active residues.reason Another reason for the lower compound 6 as a (Figure 5E,F)site thecysteine most probable forprobable this might be formation of anreactivity H-bondof between the carboxylic Michael acceptor is the pHinrange used in thisfavoring experiment. from several groups acid and S70 or R54, resulting the compound the Previous primarystudies binding pose oriented in such showed that at around pH 7, acrylic acids are negatively charged and thus, less reactive than a way that the warhead is directed away from the active site and remains unavailable fortheir binding corresponding esters analogs [44,45]. with active site cysteine residues. Another probable reason for the lower reactivity of compound 6 Quantitative determination of the rate enhancement achieved by linking the driving group with as a Michael acceptor is the pH range used in this experiment. Previous studies from several groups an acrylamide warhead was performed by one to one kinetic studies, monitored via 15N-HSQC as showed that at around pH 7, acrylic acids are negatively charged and thus, less reactive before. Kinetics measured under these conditions are more informative because only onethan leadtheir corresponding analogs compoundesters is available per [44,45]. primary binding site, which reduces the likeliness of reaction at multiple Quantitative determination of the rate enhancement achieved by linking the driving group with an acrylamide warhead was performed by one to one kinetic studies, monitored via 15 N-HSQC as before. Kinetics measured under these conditions are more informative because only one lead compound is
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available per2016, primary Molecules 21, 846 binding site, which reduces the likeliness of reaction at multiple binding 11 of 22 sites that might occur in the presence of excess compound. Compound 5 was found to be comparatively that might occur in both the presence of excess compound. Compound 5 was found to be morebinding reactivesites than compound 6 for GRXs. Evaluation of the half-time for the reaction indicated comparatively more reactive than compound 6 for both GRXs. Evaluation of the half-time for the that ester form is 30–35 fold more reactive than its acid analog. The addition of the ester modified reaction indicated that ester form isresidence 30–35 foldtime moreof reactive than its acidmoiety’s analog. The addition ofnear the the RK207 scaffold produces increased the acrylamide vinyl group ester modified RK207 scaffold produces increased residence time of the acrylamide moiety’s vinyl active site of the protein, making it more reactive than plain acrylamide [46,47]. Interestingly, this group near the active site of the protein, making it more reactive than plain acrylamide [46,47]. compound was found to be 1.5–2 fold more selective for BrmGRX (Figure 11). Interestingly, this compound was found to be 1.5–2 fold more selective for BrmGRX (Figure 11).
Figure 11. NMR signal-broadening-based kinetic studies for compounds 5 and 6 vs. naked acrylamide.
Figure 11. NMR signal-broadening-based kinetic studies for compounds 5 and 6 vs. naked acrylamide. Signal intensity of active site cysteine peaks are normalized to hydrophobic core signals using equation (2) Signal of active site are time normalized to hydrophobic signals using to intensity correct for aggregation and cysteine the loss in peaks signal over is monitored as a quantitativecore determination equation toof correct forThe aggregation andslow the for loss in signal over time is monitored as a quantitative of the(2) rate reaction. rate was very naked acrylamide and displayed a reaction half life determination reaction. The rate very for naked acrylamide displayed of about 20ofh the for rate both of proteins. Modifying thewas ester formslow of RK207 with an acrylamideand warhead a reaction half life of about h for both proteins. Modifying theproteins ester form RK207faster with an (compound 5) enhances the20rate of reaction by about 35 fold for both with aofslightly rate for warhead BrmGRX. The non-ester 5) form (compound displays a rate comparable to fold naked acrylamide. acrylamide (compound enhances the 6) rate of reaction by about 35 for both proteins with a slightly faster rate for BrmGRX. The non-ester form (compound 6) displays a rate comparable to probable explanation for this is not only the carboxylic group, but also that the RK207 nakedThe acrylamide. fragment scaffold plays some role in selectivity and is conserving this selectivity in the lead molecule. Compound 6 was found to have a rate very similar to plain acrylamide. Unfortunately, the inclusion The probable for this compound is not only the reactive carboxylic group, but also that the by RK207 of this acid formexplanation driving group makes 6 less towards BrmGRX, as suggested fragment scaffold plays some role in selectivity and is conserving this selectivity in the lead molecule. the kinetic plot in Figure 11. The reaction half time of this warhead compound for both BrmGRX and Compound 6 was found have a rate very similar plain acrylamide. Unfortunately, the inclusion hGRX was similar to to plain acrylamide. These datato suggest that the inclusion of the acid group in compound 7 does not help compound 8 in increasing reactivity with BrmGRX. of this acid form driving group makes compound 6 less reactive towards BrmGRX, as suggested by were partially corroborated by the disulfide (HED) the kineticThese plot results in Figure 11. The reaction half time of bis(2-hydroxyethyl) this warhead compound for both biological BrmGRX and assay, where some enhanced inhibition for lead compounds 5 and 6 were observed versus hGRX was similar to plain acrylamide. These data suggest that the inclusion of the acid group in naked acrylamide (Figure S23). The HED assay is a classical assay for GRXs that measures GRX compound 7 does not help compound 8 in increasing reactivity with BrmGRX. deglutathionylation activity by observing NADPH consumption at 340 nm in a reaction that couples These results were partially corroborated by the bis(2-hydroxyethyl) disulfide (HED) biological GRX and glutathione reductase. During a preincubation phase GSH and HEDS spontaneously react assay,towhere enhancedWhen inhibition lead compounds 5 and 6 were observed naked form a some heterodisulfide. GRX is for introduced it catalyzes deglutathionylation of this versus disulfide, acrylamide (Figure S23). The HED assay is a classical assay for GRXs that measures producing oxidized GSH. As quickly as it is made oxidized GSH is in turn reduced by glutathione GRX deglutathionylation byNADPH observing consumption at hands 340 nm a reaction that not couples reductase which activity consumes in aNADPH redox reaction [48]. In our theinHED assay does GRX give and completely glutathione reductase.activity Duringwith a preincubation phase GSH and HEDS reproducible the warhead reaction and we found thespontaneously concentration of react compounds needed to beWhen increased tois observe inhibition at a time scale suggested by NMRof broadening to form a heterodisulfide. GRX introduced it catalyzes deglutathionylation this disulfide, experiments. This could mean that broadening exhibited in the NMR kinetic experiments is not producing oxidized GSH. As quickly as it is made oxidized GSH is in turn reduced by glutathione completely dependent on a covalent reaction and that reduction in active site Cys signal intensity is not reductase which consumes NADPH in a redox reaction [48]. In our hands the HED assay does
give completely reproducible activity with the warhead reaction and we found the concentration of
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compounds needed to be increased to observe inhibition at a time scale suggested by NMR broadening experiments. This could mean that broadening exhibited in the NMR kinetic experiments is not completely dependent on a covalent reaction and that reduction in active site Cys signal intensity is a result of the compound occupying the active site only. In addition, a two-step serial dilution is required to titrate GRX to HED sample concentrations (10 nM) down from the stock warhead reaction (300 µM). Such a dilution destroys affinity kinetics if the compound has not yet covalently reacted and HED activity likely does not accurately reflect what’s happening in NMR samples. A more systematic approach is warranted and triplicate activity measurements taken over multiple samples will be required to statistically analyze this reaction from a biological assay perspective. Inherently weak binding of these compounds even with addition of the warhead moiety complicates the HED assay and it may prove to be more useful after additional functional group optimization of the leads affords a larger, higher affinity compound to work with. A mutagenesis approach to the HED assay as well as the NMR experiments is proposed for future work. The active site architecture of BrmGRX matches with the proposed GRX consensus active site structure which suggests the N-terminal Cys has the characteristic low pKa associated with GRXs at that residue and should be deprotonated, marking it as the likely active site Cys with warhead vinyl groups [49,50]. Since both active site Cys residues are broadened away in NMR experiments, it is impossible to know which residue is serving as the Michael donor in the warhead reaction. It is possible that both can interact at different population ratios, however covalent linking at either Cys would inhibit activity since both are required to form the disulfide bond necessary for GRX’s reaction mechanism. Nevertheless, the warhead compound may have a higher propensity to react with either Cys and therefore identification of this residue could be important for future compound affinity optimization attempts. Electrophilic warheads have recently regained researchers’ attention in the drug discovery field [3]. Previous studies have showed that in addition to their use as antineoplastics, warheads can be utilized for developing antibiotics [3,10,13]. While some risks are associated with covalent inhibitors, careful, appropriate compound selection and synthesis may result in potent antibiotic drug candidates [51]. In the search for a lead molecule that is selective towards a bacterial protein, the addition of RK207 as a moderately selective driving group for an acrylamide warhead allowed compound 5 to enhance the rate of reaction by 30–35 fold over naked acrylamide. On the other hand, compound 6 did not contribute greatly in enhancing the rate of reaction. Different functional groups present in these two compounds may orient these molecules differently while binding with BrmGRX, resulting in varying reactivity. Compound 5 seems to have a long residence time in the conserved active site of BrmGRX, leading to early completion of the reaction. Compound 6, on the other hand, orients itself in such a manner that the warhead is far away from the active site. Though these results are subtle, carefully chosen linkers with suitable synthesis schemes and several rounds of optimization may lead to a compound that is completely selective towards a bacterial protein. After scrutinizing the kinetic results, it seems that there is a need for more advanced and high throughput technology that provides more precise information about the binding mode and orientation of RK207, so that appropriate linkers can be selected and modified with it to make more effective and bacterial selective leads. In particular, NMR-guided docking will play a significant role in obtaining these poses in a high throughput manner [35]. We believe that covalent protein inhibitors have both balanced benefits and hazards. Appropriately designed experimental protocols will aid in the ultimate success of covalently bound inhibitors in drug discovery. While the selectivity for the bacterial ortholog observed in this study is rather minimal, we believe that these data indicate areas for productive future strategies. 3. Materials and Methods 3.1. General Information and Library Construction All bioanalytical grade chemicals utilized were purchased from either Thermo Fisher Scientific (Waltham, MA, USA) or Sigma Aldrich (St. Louis, MO, USA), except when otherwise noted. Warhead
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lead compounds were prepared in house. The 463-member fragment library was obtained from Maybridge (Cornwall, England) and is a subset of the Ro3 library, which has a Tanimoto similarity index between compounds of less than 0.68 [52,53]. Purity of each fragment was analyzed via 1D 1 H-NMR. Stock solutions of 100 mM or 200 mM in DMSO-d , depending on solubility, were made for 6 long term storage of each fragment. All 1D 1 H-NMR, STD-NMR, trNOE and 15 N-HSQC experiments were recorded on an Agilent DD2 750 MHz spectrometer (Agilent Technologies, Santa Clara, CA, USA) equipped with a cryoprobe, in suitable deuterated solvents and at ambient temperature. Electrospray ionization (ESI) and high-resolution mass spectra (HRMS) were taken using a Waters SyNAPT HDMS quadrupole/time-of-flight (Q/ToF) mass spectrometer (Waters, Milford, MA, USA). IR for synthesized lead compounds were taken with a Nicolet iS5 FT-IR Spectrometer (Thermo Fisher Scientific). X-ray crystallography data collected using a Bruker Kappa APEX II Duo CCD system (Bruker, Billerica, MA, USA) armed with a MO ImuS source (λ = 0.71073 Å) and a Cu Imus micro-focus source armed with QUAZAR optics (λ = 1.54178 Å) [54–56]. 3.2. Glutaredoxin Orthologs: Expression and Purification Using blastp, it was determined that among the five GRX isoforms found in Homo sapiens, hGRX1 shows the highest sequence identity with BrmGRX (38% identity) [57]. The hGRX1 isoform also contains the same conserved active (CPYC) site as the B. melitensis ortholog. BrmGRX is a 9.6 kDa protein with 88 amino acid residues whereas hGRX1 is an 11.70 kDa protein with 106 amino acid residues. Cys-to-Ser mutants of both GRX orthologs (C70S for BrmGRX and C83S for hGRX1) were prepared to enhance stability and solubility of these proteins for use at NMR concentrations. All chemical binding analysis was performed with these mutants. The expression vector containing hGRX1 was purchased from DNA2.0 as a codon optimized derivative of pJexpress 411. The expression vector for BrmGRX was provided by the Seattle Structural Genomics Center for Infectious Disease (SSGCID) as a derivative of pET28a [58]. Each vector contained a T7 polymerase inducible promotor controlling GRX expression. BL21 DE3 competent cells (New England Biolabs, Ipswich, MA, USA) were transformed with each vector respectively and cultures were raised in 15 N enriched M9 minimal media with shaking at 37 ˝ C until an OD600 of ~0.500 was attained. Temperature was then dropped to 18 ˝ C and 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to induce protein expression. Cultures were allowed to proceed with induced growth overnight before collection via centrifugation. Cell pellets were re-suspended in affinity chromatography loading buffer, lysed using a French press, and purified using immobilized metal affinity chromatography (IMAC) via a 5 mL HisTrap FF column (GE Healthcare Life Sciences, Pittsburgh, PA, USA). The loading buffer contained 20 mM Tris-Cl (pH 8.0), 200 mM NaCl, 20 mM imidazole and 2 mM dithiothreitol (DTT). Gradient buffer used to elute the purified protein contained a 20-fold higher concentration of imidazole (400 mM), but was otherwise identical in composition to loading buffer. Tobacco Etch Virus (TEV) and HRV 3C proteases were employed to cleave the 6-His tag present on the N-termini of hGRX1 and BrmGRX, respectively. Cleaved proteins were separated from free 6-His tags again using a HisTrap FF column. Purified protein was dialyzed against an NMR buffer consisting of 40 mM sodium phosphate (pH 6.5) and 2 mM DTT. Finally, samples were concentrated using an Amicon stirred cell against a 3 kDa MWCO membrane. 3.3. NMR Experiments 3.3.1. STD Screening and trNOE NMR Experiments STD and trNOE samples contained 10 µM protein, 0.5 mM fragment, 40 mM sodium phosphate buffer (pH 6.5), 2 mM DTT, and 10% deuterated glycerol. These experiments were carried out at 6.2 ˝ C. To decrease the number of samples run, each assay contained a pool of five to seven fragments of diverse scaffolds with non-overlapping 1 H peaks. All selective hits identified using this method were re-analyzed individually to verify binding with their respective GRX ortholog. Control experiments
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containing fragments only were also conducted for selective hits to verify purity. Experiments were performed using glycerol and at low temperature to counterbalance the fast tumbling rate of GRXs. These additions cause the protein to tumble slowly, resulting in retardation of rotational correlation time and therefore enhanced cross-saturation efficiency. In the STD screening experiment, a 50 ms Gaussian-shaped pulse was applied to selectively saturate the methyl protons of well-characterized proteins [20]. The following parameters were used: ´14.24 ppm off-resonance frequency, ´0.74 ppm on-resonance frequency, 0.682 s acquisition time, 2 s saturation/relaxation period, 64 scans, and 16,000 points. The efficiency of the saturation transfer by the on-resonance frequency on both proteins is shown in Supplementary Figure S6, where STD spectra of proteins only (in upfield region) are shown. To track each sample for aggregation, 1D-NMR was taken before performing STD experiments. These spectra were processed and analyzed using ACD/NMR processing software [59]. The chemical composition of the trNOE sample was consistent with STD samples. The following parameters were used for trNOE: number of points (np) was 2048 with 8 scans, relaxation delay (d1) of 2.1 s, number of increments (ni) was 80 with acquisition time of 0.128 s. The mixing time used was 0.750 s. NMRPipe and NMRFAM-SPARKY were employed for processing trNOE spectra [60,61]. Threshold values were set to parameterize STD percentages that define a fragment as a hit. To be dubbed a “hit,” the fragment must have a Rel. STD% value ě5% in the downfield region and/or ě10% in the upfield region. Rel. STD% value was calculated as: Rel.STD% “
pioff ´ ion q ˆ 100% ioff
(1)
where ioff and ion are the intensities of off-resonance and on-resonance peaks, respectively [20,21]. 3.3.2. CSP Analysis of Fragment Binding Amide resonance assignments for orthologous GRXs were retrieved from the Biological Magnetic Resonance Data Bank (BMRB) [62]. Structures of target proteins were obtained from the Protein Data Bank (PDB) [63]. The PBD code for BrmGRX is 2KHP and hGRX1 is 1JHB [19,64]. Sample composition for 15 N-HSQC experiments consists of the following: glutaredoxin (0.25 to 0.5 mM), 40 mM sodium phosphate buffer pH 6.5, 5% D2 O, 2 mM DTT, and 0–5 mM fragment. All spectra were again processed and analyzed using NMRPipe and NMRFAM-SPARKY. Kinetic studies were performed by incubating 0.3 mM of reduced protein with 0.3 mM of compound and monitoring broadening of active site cysteine peaks as a function of time as the rate of reaction. Intensity of active site cysteine peaks were normalized against a hydrophobic core glycine to correct for contributions to broadening from aggregation. 15 N-HSQC spectra were collected in succession at 25 min intervals for the first 1300 min of the reaction. Further time points were taken as instrument time was available until complete broadening away of active site peaks was observed. The percent completion of the reaction as monitored by loss of signal in the active site was normalized for aggregation using Equation (2): »´ — % Completeness “ 100 ˆ –
where,
Cys Gly
“
ICyst x IGlyt x
. In this equation
Cys max Gly
Cys max Gly
Cys tx Gly Cys max Gly
¯ fi
´
ffi fl
(2)
is the maximum peak intensity ratio of an individual active
site cysteine relative to a hydrophobic core glycine over all time points. at time point x. The peak intensity ratio is calculated as
ICyst x IGlyt x
Cys tx Gly
is the same ratio taken
where the numerator and denominator
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are the intensity of the active site cysteine being measured and the intensity of the hydrophobic core glycine respectively. G63 and G66 were used for BrmGRX and hGRX respectively. Dissociation constants (Kd ) for selected fragments were determined using titrations with increasing fragment concentrations while maintaining a constant glutaredoxin concentration. A non-linear regression (Equation (3)) was employed to calculate Kd [65,66]. Igor Pro 6.36 was used to fit the data [67]. Here, CSP(H+N) is the total Euclidean distance traveled in both hydrogen and nitrogen dimensions and is scaled by Equation (4):
CSPpH`Nq “ CSPmax
Pt ` Lt ` Kd ´
b pPt ` Lt ` Kd q2 ´ 4Lt Pt 2Pt
c ” ¯ı 1 2 ´ δ H ` α ˆ δ2N CSPpH`Nq “ 2
(3)
(4)
Values of α were 0.2 for glycines and 0.14 for all other residues [68]. CSPmax is the maximum CSP that results from saturation. Pt and Lt represents the overall concentrations of the target protein and hit compound used, respectively. Ligand efficiency (LE) was calculated using Equation (5) [69,70]: L.E. “
∆G “ ´RT ln Kd N
(5)
where, ∆G is Gibbs’ free energy, N represents the number of heavy atoms present in the hit, R is gas constant and equal to 8.314 J¨ K´1 ¨ mol´1 , T is the absolute temperature. 3.4. Computational Studies 3.4.1. Ligand Docking Prior to docking, hot spots were predicted using the FTMap web server with BrmGRX’s NMR solution structure (PDB: 2KHP) [19]. FTMap takes PDB structures as input and then samples billions of probe positions on translational and rotatable grids using the Fast Fourier Transform method. The 16 default small organic probe molecule were used. Areas on the protein surface where probes minimize and overlap are termed consensus clusters (CC) where the cluster with the largest number of probes in considered to be the primary hot spot. CCs were then extracted and superimposed onto the original apo structure. CCs that were not within reasonable proximity to residues identified by CSP analysis were removed, leaving CCs defining a large binding pocket partially occupied by GSH under physiological conditions [71] (Figure S22). Docking was conducted with the organic solvent molecules of the CCs serving to define the binding pocket, partially filling the role of a crystallographic ligand used in most site-directed docking simulations. All docking experiments were conducted and visualized within Molecular Operating Environment (MOE). A stochastic conformational library of the conjugate base form of RK207 was generated and used to sample initial binding poses. A flattened structure of RK207 was imported into MOE and partial charges were calculated before minimization in an explicit water droplet using the AMBER12-EHT force field. A conformational search was then carried out on the molecule using MOE’s automatic stochastic search function, resulting in a four conformation library used to sample initial docking poses [72]. The 2KHP structure was prepared using MOE’s automatic structure preparation after using the homology modeling tool to simulate a C70S mutation. Charges on chain termini were corrected and missing hydrogens were added before a final minimization using the same force field as above. 20,000 initial poses were sampled using triangle matcher placement, which is exhaustive for small molecules [73]. Poses were ranked using the London dG scoring function and duplicates were removed [29]. Remaining poses were refined using a rigid receptor protocol and rescored using MOE’s GBVI/WSA dG function before final duplicate removal [73]. A database of 48 final poses bound to the apo structure was generated and each model was exported to an AMBER formatted PDB file.
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3.4.2. SHIFTS Simulations for Computational CSPs Simulated proton chemical shifts were calculated using SHIFTS similarly to the McCoy and Wyss method. Only contributions to theoretical shift from ring current effects of the ligand were considered, where a ring binding at the surface of a protein can induce CSPs in atoms 7–10 Å away [31]. Nitrogen chemical shifts are not calculated due to lack of acceptable models [30,74,75]. SHIFTS calculates ring-current effects on proton shifts based on the Haigh-Maillon semi-classical model [76]. In SHIFTS simulations the benzene and pyrrole ring effects from RK207 are calculated independently of one another. McCoy and Wyss used a tryptophan based inhibitor where they set the rings of their ligand Molecules 2016, 21, 846 16 of 22 to be parameterized as a tryptophan sidechain in SHIFTS. In our method the benzene ring was set to parameters for for aa phenylalanine phenylalaninesidechain sidechainand andthe thepyrrole pyrrolering ringwas wassetset parameters from one ring toto parameters from one ring in a heme cofactor. heme pyrrole based simulation is acceptable because SHIFTS ainheme cofactor. TheThe heme pyrrole ring ring based simulation is acceptable because SHIFTS breaksbreaks apart apart cofactor rings in simulations the way samethe way the benzene and pyrrole are separated in ours. cofactor rings in simulations in the in same benzene and pyrrole are separated in ours. To compare compare simulated simulated pose pose shifts shifts to to experimental experimental data data and and filter filter out out the the dominating dominating poses, poses, the the To Pscore function (Equation (6)) proposed by Aguirrie et al. was employeed [34]. This function is P function (Equation (6)) proposed by Aguirrie et al. was employeed [34]. This function is aa score normalized version of the Q score originally used by McCoy and Wyss [31]: normalized version of the Qscore originally used by McCoy and Wyss [31]: 1 1ÿ P “= Pscore NN i
˜
(i) CSP (i) CSP piq CSP exp − CSPcalc piq CSPmax ´ CSP CSP CSPmax exp calc
¸2
(6) (6)
here, a lower Pscore indicates docked positions that are in good agreement with CSPs observed in here, a lower Pscore indicates docked positions that are in good agreement with CSPs observed in experimental data. N is the number of observable non-proline residues, CSP (i) is the experimental experimental data. N is the number of observable non-proline residues, CSPexp piq is the experimental proton CSP observed at residue i, CSP (i) is the experimental proton CSP observed at residue i, proton CSP observed at residue i, CSPcalc piq is the experimental proton CSP observed at residue and CSP and CSP maxare the largest experimental and simulated CSPs observed over all i, and CSPmax exp and CSPcalc are the largest experimental and simulated CSPs observed over all residues, respectively. residues, respectively. 3.5. Synthesis 3.5. Synthesis 3.5.1. Preparation of Methyl 4-(1H-imidazol-1-yl) Benzoate or RK464 For For the the synthesis synthesis of of RK464 RK464 (Scheme (Scheme 2), 2), aa round round bottom bottom flask flask with with aa magnetic magnetic stir stir bar bar was was used used as a reaction vessel. To this vessel, CuBr (7.2 mg, 0.025 mmol), (1R,2R)-N1,N2-dimethyl-cyclohexaneas a reaction vessel. To this vessel, CuBr (7.2 mg, 0.025 mmol), (1R,2R)-N1,N2-dimethyl- cyclohexane1,2-diamine a 1,2-diamine (16 (16 µL, μL, 0.05 0.05mmol), mmol),and andimidazole imidazole(82 (82mg, mg,0.6 0.6mmol), mmol),were wereadded addedwith withDMF DMF(20 (20mL) mL)asas solvent. with a septum and nitrogen gas was passed through it.it. a solvent.The Thevessel vesselwas wassealed sealed with a septum and nitrogen gas was passed through
Scheme 2. Synthetic or RK464. RK464. Scheme 2. Synthetic scheme scheme for for compound compound 77 or
After reacting the initial mixture of compounds, methyl 4-iodobenzoate (262 mg, 0.5 mmol) and After reacting the initial mixture of compounds, methyl 4-iodobenzoate (262 mg, 0.5 mmol) K2CO3 (357 mg, 2.6 mmol) were added. Again, nitrogen gas was back-filled and the reaction mixture and K2 CO3 (357 mg, 2.6 mmol) were added. Again, nitrogen gas was back-filled and the reaction was heated in a preheated oil bath for 48 h at constant temperature of 100 °C. Reaction progress was mixture was heated in a preheated oil bath for 48 h at constant temperature of 100 ˝ C. Reaction regularly monitored using TLC. As the reaction progressed, the color of the mixture changed from white progress was regularly monitored using TLC. As the reaction progressed, the color of the mixture to yellow. The product was extracted using ethyl acetate and purified using flash chromatography. changed from white to yellow. The product was extracted using ethyl acetate and purified using flash A pure yellow viscous product (about 40 mg) was obtained and dried under vacuum. Yield: 19%. chromatography. A pure yellow viscous product (about 40 mg) was obtained and dried under vacuum. 1H-NMR (750 MHz, CD3OD) δ: 6.75 (s, 1H, Ar-H), 6.63–6.64 (d, 2H, Ar-H), 6.19-6.20 (d, 2H, Ar-H), Yield: 19%. 1 H-NMR (750 MHz, CD3 OD) δ: 6.75 (s, 1H, Ar-H), 6.63–6.64 (d, 2H, Ar-H), 6.19-6.20 (d, 2H, 6.16 (s, 1H, Ar-H), 2.40 (s, 3H, CH3) (Supplementary Figure S9); HRMS (ESI) calcd C11H10N2O2 for Ar-H), 6.16 (s, 1H, Ar-H), 2.40 (s, 3H, CH3 ) (Supplementary Figure S9); HRMS (ESI) calcd C11 H10 N2 O2 203.0821, found 203.0808 [M+H]+, (Supplementary Figure S14). FT IR νmax cm−1: 1710.61 (carboxyl, C=O), (Supplementary Figure S20). X-ray crystallography: X-ray crystallography structure for this compound is shown in Supplementary Figure S19 with R = 0.2. 3.5.2. Preparation of 4-(1H-imidazol-1-yl)benzoic acid or RK465
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for 203.0821, found 203.0808 [M+H]+ , (Supplementary Figure S14). FT IR νmax cm´1 : 1710.61 (carboxyl, C=O), (Supplementary Figure S20). X-ray crystallography: X-ray crystallography structure for this compound is shown in Supplementary Figure S19 with R = 0.2. 3.5.2. Preparation of 4-(1H-imidazol-1-yl)benzoic acid or RK465 In a round-bottom flask, 20 mg of compound 7 was dissolved in 4 mL of ethanol and stirred with 2 mL of 5% NaOH solution for 2 h (Scheme 3). Formation of the hydrolyzed product was regularly monitored Molecules 2016,using 21, 846TLC. 17 of 22
Scheme Scheme 3. 3. Synthetic Synthetic scheme scheme for for compound compound 88 or or RK465. RK465.
After 2 h, the reaction appeared close to completion as suggested by TLC. At this point, 1 M HCl After 2 h, the reaction appeared close to completion as suggested by TLC. At this point, 1 M HCl was added dropwise until the mixture turned acidic (monitored with litmus paper). A white product was added dropwise until the mixture turned acidic (monitored with litmus paper). A white product precipitated after roto-vaporization. The product was soluble in methanol, leaving behind NaCl precipitated after roto-vaporization. The product was soluble in methanol, leaving behind NaCl crystals. The product was dried under vacuum, resulting in a white powder. Yield: 90%. 11H-NMR crystals. The product was dried under vacuum, resulting in a white powder. Yield: 90%. H-NMR (750 MHz, CD3OD) δ 9.61 (s, 1H, Ar-H), 8.25–8.26 (d, 2H, Ar-H), 8.18 (s, 1H, Ar-H), 7.88-7.90 (d, 2H, (750 MHz, CD3 OD) δ 9.61 (s, 1H, Ar-H), 8.25–8.26 (d, 2H, Ar-H), 8.18 (s, 1H, Ar-H), 7.88-7.90 (d, 2H, Ar-H), 7.82 (s, 1H, Ar-H), (Supplementary Figure S10). FT IR νmax cm−1: 1715.44 (carboxyl, C=O), Ar-H), 7.82 (s, 1H, Ar-H), (Supplementary Figure S10). FT IR νmax cm´1 : 1715.44 (carboxyl, C=O), (Supplementary Figure S21). HRMS: calculated for C10H8N2O2 189.0664, found 189.0672 [M + H]+, (Supplementary Figure S21). HRMS: calculated for C10 H8 N2 O2 189.0664, found 189.0672 [M + H]+ , (Supplementary Figure S15). (Supplementary Figure S15). 3.5.3. (4) 3.5.3. Preparation Preparation of of 4-(3-(Acrylamidomethyl)-1H-pyrrol-1-yl)benzoate 4-(3-(Acrylamidomethyl)-1H-pyrrol-1-yl)benzoate (4) To round bottom bottom flask flask was was added added CuBr CuBr (7.2 (7.2 mg), mg), (1R,2R)-N1,N2-dimethyl(1R,2R)-N1,N2-dimethyl- cyclohexane-1,2To aa round cyclohexane-1,2diamine (16 μL), and (1H-pyrrol-3-yl) methanamine (58 mg). diamine (16 µL), and (1H-pyrrol-3-yl) methanamine (58 mg). The The mixture mixture was was sealed sealed with with septum, septum, evacuated, and back-filled with nitrogen gas. Methyl 4-iodobenzoate (262 mg) and K 2CO3 (357 mg) evacuated, and back-filled with nitrogen gas. Methyl 4-iodobenzoate (262 mg) and K2 CO3 (357 mg) were then mL) was used as aassolvent. Sodium carbonate was preferred because using were then added. added.DMF DMF(20 (20 mL) was used a solvent. Sodium carbonate was preferred because potassium hydroxide (a hard base) resulted in hydrolysis of the ester moiety of the product. The using potassium hydroxide (a hard base) resulted in hydrolysis of the ester moiety of the product. ligand to copper ratio was 2:1. The mixture was heated for 2 days at 100 °C. Reaction progress was The ligand to copper ratio was 2:1. The mixture was heated for 2 days at 100 ˝ C. Reaction progress monitored via TLC. EthylEthyl acetate was used to extract the organic phasephase from the aqueous layer. Flash was monitored via TLC. acetate was used to extract the organic from the aqueous layer. chromatography was used to purify the product. The white-colored product was eluted with a Flash chromatography was used to purify the product. The white-colored product was eluted with 1 mixture ofof ethyl acetate to hexane with a yield of 20%. H-NMR (750 (750 MHz,MHz, CDClCDCl 3) δ 8.37 (s, 1H, NH), 1 H-NMR a mixture ethyl acetate to hexane with a yield of 20%. 3 ) δ 8.37 (s, 1H, 7.85–7.86 (d, 2H, Ar-H), 7.11 (s, 1H, Ar-H), 6.76 (s, 1H, Ar-H), 6.58–6.59 (d, 2H, Ar-H), (s, 1H, NH), 7.85–7.86 (d, 2H, Ar-H), 7.11 (s, 1H, Ar-H), 6.76 (s, 1H, Ar-H), 6.58–6.59 (d, 2H,6.22 Ar-H), 6.22Ar(s, H), 4.23 (s, 2H, CH 2, 3.84 (s, 3H, CH3), (Supplementary Figure S11). HRMS: calculated for C12H12N2O2 1H, Ar-H), 4.23 (s, 2H, CH2 , 3.84 (s, 3H, CH3 ), (Supplementary Figure S11). HRMS: calculated for 253.0953, 253.0936 [M +253.0936 Na]+, (Supplementary Figure S16). Figure S16). C H N found O 253.0953, found [M + Na]+ , (Supplementary 12
12
2
2
3.5.4. Preparation of Methyl 4-(3-(Acrylamidomethyl)-1H-pyrrol-1-yl)benzoate (5) In a round round bottom bottom flask, flask, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b] 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b] pyridinium 3-oxid hexafluorophosphate hexafluorophosphate(HATU, (HATU,102 102 mg), triethylamine 40 μL), acrylic (12were μL) mg), triethylamine (Et3(Et N,3N, 40 µL), andand acrylic acid acid (12 µL) were with (5 DMF mL). The mixture was with sealed with septum, evacuated, and back-filled with taken taken with DMF mL).(5The mixture was sealed septum, evacuated, and back-filled with nitrogen nitrogen After3015mg min, 30 mg of compound 4 was added to theAgain, mixture. theback-filled flask was gas. Aftergas. 15 min, of compound 4 was added to the mixture. the Again, flask was back-filled with gasstirred for 1 hfor and stirred fortemperature. 48 h at roomAtemperature. A yellowish with nitrogen gasnitrogen for 1 h and 48 h at room yellowish solid compoundsolid was 1 1 compound was formed with aH-NMR yield of (750 23%.MHz, H-NMR (750 MHz, CDCl 3)NH), δ: 8.63 (s, 1H, NH), 8.08–8.09 formed with a yield of 23%. CDCl ) δ: 8.63 (s, 1H, 8.08–8.09 (d, 2H, Ar-H), 3 (d, 7.87 (s, 1H, Ar-H), 7.40–7.41 (d,7.12 2H,(s, Ar-H), 7.12 (s, 6.78–6.79 1H, Ar-H),(d, 6.78–6.79 (d, 1H, 6.59–6.63 -C=CH-), 7.872H, (s, Ar-H), 1H, Ar-H), 7.40–7.41 (d, 2H, Ar-H), 1H, Ar-H), 1H, -C=CH-), 6.59–6.63 (m, 2H, Ar-H & H2C=C-), 6.36 (s, 1H, H2C=C-), 4.28 (s, 2H, CH2), 3.92 (s, 3H, CH3) (Supplementary Figure S12). HRMS: calculated for C16H16N2O3 307.1059, found 307.1058 [M + Na]+ (Supplementary Figure S17A,B). 3.5.5. Preparation of 4-(3-(Acrylamidomethyl)-1H-pyrrol-1-yl)benzoic acid (6)
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(m, 2H, Ar-H & H2 C=C-), 6.36 (s, 1H, H2 C=C-), 4.28 (s, 2H, CH2 ), 3.92 (s, 3H, CH3 ) (Supplementary Figure S12). HRMS: calculated for C16 H16 N2 O3 307.1059, found 307.1058 [M + Na]+ (Supplementary Figure S17A,B). 3.5.5. Preparation of 4-(3-(Acrylamidomethyl)-1H-pyrrol-1-yl)benzoic acid (6) Compound 5 (10 mg) was hydrolyzed to form the acid derivative 6. The mixture was dissolved in 3 mL of acetone and stirred with 2 mL of 5% NaOH for 3 h. Formation of product was monitored with TLC. HCl (1 M) was added dropwise until an acidic pH was reached. A white precipitate was formed and purified via filtration. 1 H-NMR (750 MHz, CDCl3 ) δ: 8.02 (s, 1H, NH), 7.85–7.90 (d, 2H, Ar-H), 7.39 (s, 1H, Ar-H), 7.22–7.28 (d, 2H, Ar-H), 7.11 (s, 1H, Ar-H), 6.80–6.81 (d, 1H, -C=CH-), 6.61–6.65 (m, 2H, Ar-H & H2 C=C- ), 6.50 (s, 1H, H2 C=C-), 4.31 (s, 2H, CH2 ) (Supplementary Figure S13). HRMS: calculated for C15 H14 N2 O3 269.0926, found 269.0911 [M ´ H]´ (Supplementary Figure S18). 3.6. HED Assay Warhead mediated inhibition of GRX activity was monitored using the HED assay, originally proposed by Nagai and Black [77]. The protocol here was adapted from Zaffagnini et al. with the only modification of doubling the concentration of glutathione reductase (GR) [78]. This was done because we experienced GR being a rate limiting reagent when attempting to run trials with GRX concentrations above 10 nM. Briefly, a reaction mixture containing 0.7 mM HEDS, 0.1 mg/mL BSA, 1 mM reduced GSH, 12 µg/mL glutathione reductase, and 0.2 mM NADPH was assembled in 100 mM Tris-Cl, pH 7.9. The reaction mixture is separated equally into a sample and reference cuvette and after a 3-minute incubation to facilitate the spontaneous formation of the GSH-HED mixed dosulfide, 10 nM GRX is added to the sample cuvette alongside an equal amount of buffer to the sample cuvette. Activity was monitored by the GR catalyzed oxidation of NADPH at 340 nm as it reduces the oxidized glutathione produced by GRX using a Shimadzu UV-1800 spectrophotometer. GRX activity is isolated by observing the change in absorbance at 340 nm in the sample cuvette versus the reference cuvette as a subtraction from the baseline NADPH oxidation rate. GRX activity was calculated from the first 60 s of each reaction and activity was expressed at mM of NADPH oxidized/min. Sample preparation for the HED assays was slightly different compared with set-up for the NMR experiments. Initial attempts at a 1:1 stoichiometric ratio of compound to protein produced little reduction in activity in early time points. In an effort to speed up the reaction and see inhibition at the beginning of the reaction a ratio of 4:1 compound to protein was selected moving forward. This requires a compound concentration of 1.2 mM which unfortunately was not soluble under 100% aqueous conditions. Therefore, stock reactions for the HEDS assay were set up with 12% DMSO to facilitate solubility of the compounds at 1.2 mM. Otherwise the reactions were set up the same as NME kinetic studies with 40 mM sodium-phosphate buffer at pH 6.5, 20 mM NaCl, 0.3 mM GRX, and 1.2 mM compound. For Figure S20, four reactions were started at the same time with compound 5, compound 6, acrylamide, and an apo reaction to which only DMSO was added to 12%. Activity was taken twice a day for 4 days. 4. Conclusions This study presents the design, synthesis and development of a covalent inhibitor for a bacterial protein using the principles of FBDD. A small library was screened and validated with NMR methods for two GRX orthologs. Most of the hits obtained were found to show low millimolar selectivity for the bacterial GRX. A majority of the hits were versatile in nature, binding with both GRXs. The best hit (RK207), was submitted to an NMR guided docking routine to elucidate fragment binding poses. An ester modification of this RK207 driving group coupled with an acrylamide warhead enhanced the rate of reaction by 30 to 35-fold with small selectivity for the bacterial protein, while the acid form of this compound does not enhance reactivity. These results may inspire future projects to
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develop more selective covalent inhibitors for bacterial GRXs by optimizing the driving group with an appropriate linker. Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/ 7/846/s1. Acknowledgments: This study was financially supported by The University of Akron Office or Research, which provided start-up funds to TCL. We wish to thank the Kresge Foundation and donors to the Kresge Challenge Program at The University of Akron for funds used to purchase the NMR instrument used in this work. The National Science Foundation (CHE-0840446) for funds used to purchase the Bruker APEX II Duo X-ray diffractometer used in this research. We would like to thank Erendra Manandhar, Joel Caporoso, Mahesh Dawadi, Deepak Koirala, Bishnu Prasad Thapaliya, Louis Ray, Selim Gerislioglu, and the NMR facility faculty of “The University of Akron” for their help and guidance to complete this work. Finally, we would like to thank David, case for his assistance in customizing SHIFTS for this work. Author Contributions: R.K., D.M., C.D., A.C., S.B., M.P., and M.D. carried out experimental work. R.K., D.M., and A.C. prepared and edited the manuscript. T.L. provided inspiration and experimental design for the project, supervised the experimental work of the authors, and edited the manuscript. Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations The following abbreviations are used in this manuscript: FBDD STD Relative STD% NMR HSQC SAR BrmGRX hGRX1 E. coli HTS log P Kd L.E. CSPs trNOE DMSO D2 O DTT NaCl Et3N HATU
fragment-based drug discovery saturation transfer difference relative saturation transfer difference in percentage nuclear magnetic resonance heteronuclear single quantum correlation structure activity relationship Brucella melitensis glutaredoxin human glutaredoxin 1 Escherichia coli high throughput screening log of partition-coefficient dissociation constant ligand efficiency chemical shift perturbations transferred nuclear Overhauser effect dimethyl sulfoxide deuterium oxide dithiothreitol sodium chloride triethyl amine 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate
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